The vascular endothelium is a selective cellular barrier to circulatory proteins and is critical to vessel wall homeostasis. Increased endothelial cell permeability occurs in acute lung injury resulting in profound physiologic derangement. Contractile proteins, actin and myosin, control permeability and bioactive agonists such as thrombin induce endothelial cell contraction, intercellular gap formation and barrier dysfunction by signaling changes in actomyosin. We have shown that thrombin receptor proteolysis is sequentially coupled to Ca2+ (i) increases, protein kinase C (PKC) activation and myosin light chain (MLC20), phosphorylation catalyzed by a novel myosin light chain kinase. These events result in loss of endothelial cell monolayer resistance, paracellular gap formation and ultimately barrier dysfunction. In Project #2 we will test the hypothesis that myosin-specific phosphatases (PPases) determine the phosphorylation status of myosin and are key to regulating the endothelial cell contractile state. Although several PPases can dephosphorylate isolated MLC, myosin-associated PPase properties in endothelium are unknown. Our preliminary data suggests that both a thrombin-inducible Ca2+/calmodulin (CaM)-dependent PPase (2B) and a constitutively active myosin-associated PPase 1 may be involved in MLC dephosphorylation in endothelium. In this proposal we will explore the role of myosin PPase(s) in the regulation of endothelial cell contraction/relaxation and barrier function in both human and bovine endothelium. SA #1 will utilize standard biochemical, molecular and immunofluorescent techniques to more clearly define myosin-associated constitutive PPase 1 catalytic and regulatory subunits and activity in endothelium. SA #2 will identify thrombin-inducible PPase activities in endothelium by examination of basal and agonist-stimulated myosin-specific and nonspecific PPase activities and the effect of semi-selective and specific PPase inhibitors on endothelial cell PPase activity, MLC phosphorylation, gap formation and barrier function. Finally, thrombin- mediated MLC20 phosphorylation, contraction and barrier dysfunction are critically dependent upon PKC activity and significantly attenuated (barrier protection) by cAMP via activation of its target protein kinase A (PKA). We will investigate the role of cAMP/PKA (SA #3) and PKC (SA #4) in the regulation of myosin-specific PPase activity by determining the effect of specific PKA/PKC inhibitors and activators on PPase activity associated with contractile apparatus. To further examine PKC regulation of endothelial cell PPase(s) we will utilize specific inhibitors/activators and by selective isozyme-specific PKC depletion or overexpression. These studies which define the complex regulation of myosin phosphorylation status will elucidate novel levels of endothelial contractile regulation and a better understanding of the vascular permeability processes which accompany lung inflammation.